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Leukemia cells are highly dependent on glucose and glutamine as bioenergetic and biosynthetic fuels. Inhibition of the metabolism of glucose but also of glutamine is thus proposed as a therapeutic modality to block leukemia cell growth. Since glucose also supports protein glycosylation, we wondered whether part of the growth inhibitory effects resulting from glycolysis inhibition could indirectly result from a defect in glycosylation of glutamine transporters. We found that ASCT2/SLC1A5, a major glutamine transporter, was indeed deglycosylated upon glucose deprivation and 2-deoxyglucose exposure in HL-60 and K-562 leukemia cells. Inhibition of glycosylation by these modalities as well as by the bona fide glycosylation inhibitor tunicamycin however marginally influenced glutamine transport and did not impact on ASCT2 subcellular location. This work eventually unraveled the dispensability of ASCT2 to support HL-60 and K-562 leukemia cell growth and identified the upregulation of the neutral amino acid antiporter LAT1/SLC7A5 as a mechanism counteracting the inhibition of glycosylation. Pharmacological inhibition of LAT1 increased the growth inhibitory effects and the inactivation of the mTOR pathway resulting from glycosylation defects, an effect further emphasized during the regrowth period post-treatment with tunicamycin. In conclusion, this study points towards the underestimated impact of glycosylation inhibition in the interpretation of metabolic alterations resulting from glycolysis inhibition, and identifies LAT1 as a therapeutic target to prevent compensatory mechanisms induced by alterations in the glycosylating process., Competing Interests: The authors declare no competing interest.

Transgenic and gene knockout rodent models are primordial to study pathophysiological processes in cardiovascular research. Over time, cardiac MRI has become a gold standard for in vivo evaluation of such models. Technical advances have led to the development of magnets with increasingly high field strength, allowing specific investigation of cardiac anatomy, global and regional function, viability, perfusion or vascular parameters. The aim of this report is to provide a review of the various sequences and techniques available to image mice on 7-11.7 T magnets and relevant to the clinical setting in humans. Specific technical aspects due to the rise of the magnetic field are also discussed.

Introduction: Radiolabeled antibodies directed against endoglin (CD105) are promising tools for imaging and antiangiogenic cancer therapy. To validate iodinated antibodies as reliable tracers, we investigated the influence of the radiolabeling method (direct or indirect) on their in vivo stability., Methods: Anti-CD105 mAbs were radioiodinated directly using chloramine-T ((125)I-anti-CD105-mAbs) or indirectly using D-KRYRR peptide as a linker ((125)I-KRYRR-anti-CD105-mAbs). The biodistribution was studied in B16 tumor-bearing mice via SPECT/CT imaging., Results: Radioiodinated mAbs were stable in vitro. In vivo, thyroid showed the most important increase of uptake after 24h for (125)I-anti-CD105-mAbs (91.9±4.0%ID/ml) versus(125)I-KRYRR-anti-CD105-mAbs (4.4±0.6%ID/ml). Tumor uptake of (125)I-anti-CD105-mAbs (0.9±0.3%ID/ml) was significantly lower than that of (125)I-KRYRR-anti-CD105-mAbs (4.7±0.2%ID/ml)., Conclusions: An accurate characterization of the in vivo stability of radioiodinated mAbs and the choice of an appropriate method for the radioiodination are required, especially for novel targets. The indirect radioiodination of internalizing anti-CD105 mAbs leads to more stable tracer by decreasing in vivo deiodination and improves the tumor retention of radioiodinated mAbs., Advances in Knowledge and Implications for Patient Care: To date, the only antiangiogenic antibody approved for clinical indications is bevacizumab. There is a need to develop more antibodies that have targets highly expressed on tumor endothelium. CD105 represents a promising marker of angiogenesis, but its therapeutic relevance in cancer needs to be further investigated. In this context, this study suggests the potential use of indirectly iodinated anti-CD105 mAbs for tumor imaging and for therapeutic purposes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3 μM, 1 hour) and chronic (10 mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.

PEGylated chitosan-based nanoparticles offer attractive platforms for siRNA cocktail delivery into tumors. Still, therapeutic efficacy requires us to select a rational combination of siRNAs and an efficient tumor delivery after systemic administration. Here, we showed that non-covalent PEGylation of chitosan-based nanoparticles loaded with siRNA targeting two key transporters of energy fuels for cancer cells, namely the lactate transporter MCT1 and the glutamine transporter ASCT2, could lead to significant antitumor effects. As a ligand, we tested variations of the prototypical RGD peptidomimetic (RGDp). A higher siRNA delivery was obtained with naphthyridine-containing RGDp randomly conjugated on the PEG chain by clip photochemistry and the use of a lipophilic linker than when using traditional chain-end grafting and RGDp with a hydrophilic linker. The antiproliferative effects resulting from ASCT2 and MCT1 silencing were validated separately in vitro in conditions mimicking specific metabolic profiles of cancer cells and in vivo upon concomitant delivery. The combination of those siRNA and the selected components of targeted RGDp nanoparticles led to a dramatic tumor growth inhibition upon peri-tumoral but also systemic administration in mice. Altogether these data emphasize the convenience of using non-covalent PEGylated chitosan particles to produce sheddable stealth protection compatible with an efficient siRNA delivery in tumors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Leukemia cells are described as a prototype of glucose-consuming cells with a high turnover rate. The role of glutamine in fueling the tricarboxylic acid cycle of leukemia cells was however recently identified confirming its status of major anaplerotic precursor in solid tumors. Here we examined whether glutamine metabolism could represent a therapeutic target in leukemia cells and whether resistance to this strategy could arise. We found that glutamine deprivation inhibited leukemia cell growth but also led to a glucose-independent adaptation maintaining cell survival. A proteomic study revealed that glutamine withdrawal induced the upregulation of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT), two enzymes of the serine pathway. We further documented that both exogenous and endogenous serine were critical for leukemia cell growth and contributed to cell regrowth following glutamine deprivation. Increase in oxidative stress upon inhibition of glutamine metabolism was identified as the trigger of the upregulation of PHGDH. Finally, we showed that PHGDH silencing in vitro and the use of serine-free diet in vivo inhibited leukemia cell growth, an effect further increased when glutamine metabolism was blocked. In conclusion, this study identified serine as a key pro-survival actor that needs to be handled to sensitize leukemia cells to glutamine-targeting modalities.

Recently, a new generation of highly promising inhibitors bearing β-keto-enol functionality has emerged. Reported herein is the first synthesis and use of novel designed drugs based on the β-keto-enol group embedded with heterocyclic moieties such as pyrazole, pyridine, and furan, prepared in a one-step procedure by mixed Claisen condensation. All the newly synthesized compounds were characterized by FT-IR, ¹H-NMR, (13)C-NMR, ESI/LC-MS, elemental analysis, and evaluated for their in vitro antiproliferative activity against breast cancer (MDA-MB241) human cell lines and fungal strains (Fusarium oxysporum f.sp albedinis FAO). Three of the synthesized compounds showed potent activity against fungal strains with IC50 values in the range of 0.055-0.092 µM. The results revealed that these compounds showed better IC50 values while compared with positive controls.

We investigated the impact of oxygenation status (measured by EPR oximetry) on the uptake of (18)F-FDG (measured by PET) in two different tumor models during a carbogen breathing challenge. We observed a significant drop in (18)F-FDG uptake under carbogen breathing that suggests a rapid metabolic adaptation to the oxygen environment., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvβ3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvβ3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We studied intraobserver (n = 24), interobserver (n = 24) and interexperiment (n = 12) reproducibility of left ventricular (LV) mass and volume measurements in mice using an 11.7 T MRI system. The LV systolic function was assessed with a short-axis FLASH-cine sequence in 29 mice, including animals having undergone transverse aortic constriction. Bland-Altman and regression analysis were used to compare the different data sets. Reproducibility was excellent for the LV mass and end-diastolic volume (coefficient of variability (CoV) between 5.4 and 11.8 %), good for end-systolic volume (CoV 15.2-19.4 %) and moderate for stroke volume and ejection fraction (CoV 14.7-20.9 %). We found an excellent correlation between LV mass determined by MRI and ex vivo morphometric data (r = 0.92). In conclusion, LV systolic function can be assessed on an 11.7 T MRI scanner with high reproducibility for most parameters, as needed in longitudinal studies. However, data should be interpreted taking into account the moderate reproducibility of small volumes.

Purpose of Review: Glutamine and acetate were recently identified as alternatives to glucose for fueling the tricarboxylic acid (TCA) cycle in cancer cells, particularly in the context of hypoxia., Recent Findings: Molecular mechanisms orchestrating glutamine and acetate metabolism were elicited through the combination of C tracer analysis and genetic silencing, or pharmacological modulation of key metabolic enzymes including those converting glutamate into α-ketoglutarate (αKG) (and beyond) and acetate into acetyl-coenzyme A (CoA)., Summary: Oxidative decarboxylation and reductive carboxylation of αKG represent two options for the glutamine metabolism. The canonical forward mode of the TCA cycle fuelled by glutamine may benefit from the decarboxylation of malate into pyruvate for fueling pyruvate dehydrogenase and generating acetyl-CoA to offer a self-sustainable TCA cycle. Under hypoxia and mutations in the TCA cycle, the reductive carboxylation of glutamine-derived αKG into citrate mainly supports lipogenesis via the ATP citrate lyase that cleaves citrate into oxaloacetate and acetyl-CoA. Still, a largely unsuspected source of acetyl-CoA was shown to derive from the direct ligation of acetate to CoA by acetyl-CoA synthetases. Altogether, these findings identify critical metabolic nodes in the glutamine and acetate metabolism as new determinants of tumor metabolic plasticity that may facilitate the design of synthetic lethal treatments.

Cell tracking could be useful to elucidate fundamental processes of cancer biology such as metastasis. The aim of this study was to visualize, using MRI, and to quantify, using electron paramagnetic resonance (EPR), the entrapment of murine breast cancer cells labeled with superparamagnetic iron oxide particles (SPIOs) in the mouse brain after intracardiac injection. For this purpose, luciferase-expressing murine 4 T1-luc breast cancer cells were labeled with fluorescent Molday ION Rhodamine B SPIOs. Following intracardiac injection, SPIO-labeled 4 T1-luc cells were imaged using multiple gradient-echo sequences. Ex vivo iron oxide quantification in the mouse brain was performed using EPR (9 GHz). The long-term fate of 4 T1-luc cells after injection was characterized using bioluminescence imaging (BLI), brain MRI and immunofluorescence. We observed hypointense spots due to SPIO-labeled cells in the mouse brain 4 h after injection on T2 *-weighted images. Histology studies showed that SPIO-labeled cancer cells were localized within blood vessels shortly after delivery. Ex vivo quantification of SPIOs showed that less than 1% of the injected cells were taken up by the mouse brain after injection. MRI experiments did not reveal the development of macrometastases in the mouse brain several days after injection, but immunofluorescence studies demonstrated that these cells found in the brain established micrometastases. Concerning the metastatic patterns of 4 T1-luc cells, an EPR biodistribution study demonstrated that SPIO-labeled 4 T1-luc cells were also entrapped in the lungs of mice after intracardiac injection. BLI performed 6 days after injection of 4 T1-luc cells showed that this cell line formed macrometastases in the lungs and in the bones. Conclusively, EPR and MRI were found to be complementary for cell tracking applications. MRI cell tracking at 11.7 T allowed sensitive detection of isolated SPIO-labeled cells in the mouse brain, whereas EPR allowed the assessment of the number of SPIO-labeled cells in organs shortly after injection., (Copyright © 2015 John Wiley & Sons, Ltd.)

Background: The Warburg phenotype identified decades ago describes tumor cells with increased glycolysis and decreased mitochondrial respiration even in the presence of oxygen. This particular metabolism also termed 'aerobic glycolysis' reflects an adaptation of tumor cells to proliferation in a heterogeneous tumor microenvironment. Although metabolic alterations in cancer cells are common features, their impact on the response to radiotherapy is not yet fully elucidated. This study investigated the impact of cellular oxygen consumption inhibition on the tumor response to radiotherapy., Material and Methods: Warburg-phenotype tumor cells with impaired mitochondrial respiration (MD) were produced and compared in respect to their metabolism to the genetically matched parental cells (WT). After characterization of their metabolism we compared the response of MD cells to irradiation in vivo and in vitro to the genetically matched parental cells (WT)., Results: We first confirmed that MD cells were exclusively glycolytic while WT cells exhibited mitochondrial respiration. We then used these cells for assessing the response of WT and MD tumors to a single dose of radiation and showed that the in vivo tumor growth delay of the MD group was increased, indicating an increased radiosensitivity compared to WT while the in vitro ability of both cell lines to repair radiation-induced DNA damage was similar., Conclusion: Taken together, these results indicate that in addition to intrinsic radiosensitivity parameters the tumor response to radiation will also depend on their metabolic rate of oxygen consumption.

Abnormal architecture of the tumor blood network, as well as heterogeneous erythrocyte flow, leads to temporal fluctuations in tissue oxygen tension exposing tumor and stromal cells to cycling hypoxia. Inflammation is another feature of tumor microenvironment and is considered as a new enabling characteristic of tumor progression. As cycling hypoxia is known to participate in tumor aggressiveness, the purpose of this study was to evaluate its role in tumor-promoting inflammation. Firstly, we assessed the impact of cycling hypoxia in vitro on endothelial inflammatory response induced by tumor necrosis factor α. Results showed that endothelial cells exposed to cycling hypoxia displayed an amplified proinflammatory phenotype, characterized by an increased expression of inflammatory cytokines, namely, interleukin (IL)-6 and IL-8; by an increased expression of adhesion molecules, in particular intercellular adhesion molecule-1 (ICAM-1); and consequently by an increase in THP-1 monocyte adhesion. This exacerbation of endothelial inflammatory phenotype occurs through nuclear factor-κB overactivation. Secondly, the role of cycling hypoxia was studied on overall tumor inflammation in vivo in tumor-bearing mice. Results showed that cycling hypoxia led to an enhanced inflammation in tumors as prostaglandin-endoperoxide synthase 2 (PTGS2), IL-6, CXCL1 (C-X-C motif ligand 1), and macrophage inflammatory protein 2 (murine IL-8 functional homologs) mRNA expression was increased and as a higher leukocyte infiltration was evidenced. Furthermore, cycling hypoxia-specific inflammatory phenotype, characterized by a simultaneous (baculoviral inhibitor of apoptosis repeat-containing 5)(low)/PTGS2(high)/ICAM-1(high)/IL-6(high)/IL-8(high) expression, is associated with a poor prognosis in human colon cancer. This new phenotype could thus be used in clinic to more precisely define prognosis for colon cancer patients. In conclusion, our findings evidenced for the first time the involvement of cycling hypoxia in tumor-promoting inflammation amplification., (Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to develop vitamin E-based micelles loaded with Doxorubicin (DOX) (DOX-TOS-TPGS), taking advantages of the anti-cancer activity of vitamin E derivatives: Tocopherol succinate (TOS) and D-α-tocopherol polyethylene2000 succinate (TPGS). Therefore, we developed micelles consisting in a mixture of TOS (as solubilizer) and TPGS2000 (as stabilizer) (1:1). DOX-TOS-TPGS micelles exhibited a size of 78 nm and a ζ potential of -7 mV. High drug loading (40% w/w) was achieved. The critical micellar concentration was determined at 14 μg/ml. In vitro, after 24 h, DOX-TOS-TPGS micelles exhibited higher cytotoxicity than free-DOX (IC50 on MCF-7 cells, at 24 h, 58 vs 5 μg/ml). In vivo anti-tumor efficacy, performed on two tumor models (CT26 and MCF-7), demonstrated a 100% long-term survival of mice when treated with DOX-TOS-TPGS compared to DOX-free. Interestingly, the survival time of mice treated with unloaded TOS-TPGS micelles was similar to DOX-free, indicating an anti-cancer activity of vitamin E derivatives. Based on these results, it can be concluded that the formulations developed in this work may be considered as an effective DOX delivery system for cancer chemotherapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Extracellular tumor acidosis largely results from an exacerbated glycolytic flux in cancer and cancer-associated cells. Conversely, little is known about how tumor cells adapt their metabolism to acidosis. Here, we demonstrate that long-term exposure of cancer cells to acidic pH leads to a metabolic reprogramming toward glutamine metabolism. This switch is triggered by the need to reduce the production of protons from glycolysis and further maintained by the NAD(+)-dependent increase in SIRT1 deacetylase activity to ensure intracellular pH homeostasis. A consecutive increase in HIF2α activity promotes the expression of various transporters and enzymes supporting the reductive and oxidative glutamine metabolism, whereas a reduction in functional HIF1α expression consolidates the inhibition of glycolysis. Finally, in vitro and in vivo experiments document that acidosis accounts for a net increase in tumor sensitivity to inhibitors of SIRT1 and glutaminase GLS1. These findings highlight the influence that tumor acidosis and metabolism exert on each other., (©2014 American Association for Cancer Research.)

Aims: Antibody-labeled gold nanoparticles represent an attractive tool for cancer imaging and therapy. In this study, the anti-CD105 antibody was conjugated with gold nanoparticles (AuNPs) for the first time. The antibody biodistribution in mice before and after conjugation to AuNPs was studied, with a focus on tumor targeting., Materials & Methods: Antibodies were radiolabeled with 89Zr before conjugation to AuNPs (5 nm). Immunonanoconjugates were characterized in vitro in terms of size, stability in plasma and binding to the target. Quantitative PET imaging and ICP-MS analysis assessed in vivo distribution and specific tumor targeting of tracers., Results: The tumor uptake of immunoconjugates was preserved up to 24 h after injection, with high tumor contrast and selective tumor targeting. No major tracer accumulation was observed over time in nonspecific organs. ICP-MS analysis confirmed the antibody specificity after nanoparticle conjugation., Conclusion: The anti-CD105 antibody conjugation to AuNPs did not greatly affect CD105-dependent tumor uptake and the efficacy of tumor targeting for cancer detection.

Introduction: Stem cells from the apical papilla (SCAP) are a population of mesenchymal stem cells likely involved in regenerative endodontic procedures and have potential use as therapeutic agents in other tissues. In these situations, SCAP are exposed to hypoxic conditions either within a root canal devoid of an adequate blood supply or in a scaffold material immediately after implantation. However, the effect of hypoxia on SCAP proliferation and differentiation is largely unknown. Therefore, the objective of this study was to evaluate the effect of hypoxia on the fate of SCAP., Methods: SCAP were cultured under normoxia (21% O2) or hypoxia (1% O2) in basal or differentiation media. Cellular proliferation, gene expression, differentiation, and protein secretion were analyzed by live imaging, quantitative reverse-transcriptase polymerase chain reaction, cellular staining, and enzyme-linked immunosorbent assay, respectively., Results: Hypoxia had no effect on SCAP proliferation, but it evoked the up-regulation of genes specific for osteogenic differentiation (runt-related transcription factor 2, alkaline phosphatase, and transforming growth factor-β1), neuronal differentiation ( 2'-3'-cyclic nucleotide 3' phosphodiesterase, SNAIL, neuronspecific enolase, glial cell-derived neurotrophic factor and neurotrophin 3), and angiogenesis (vascular endothelial growth factor A and B). Hypoxia also increased the sustained production of VEGFa by SCAP. Moreover, hypoxia augmented the neuronal differentiation of SCAP in the presence of differentiation exogenous factors as detected by the up-regulation of NSE, VEGFB, and GDNF and the expression of neuronal markers (PanF and NeuN)., Conclusions: This study shows that hypoxia induces spontaneous differentiation of SCAP into osteogenic and neurogenic lineages while maintaining the release of the proangiogenic factor VEGFa. This highlights the potential of SCAP to promote pulp-dentin regeneration. Moreover, SCAP may represent potential therapeutic agents for neurodegenerative conditions because of their robust differentiation potential., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: Temporal and local fluctuations in O2 in tumors require adaptive mechanisms to support cancer cell survival and proliferation. The transcriptome associated with cycling hypoxia (CycHyp) could thus represent a prognostic biomarker of cancer progression., Method: We exposed 20 tumor cell lines to repeated periods of hypoxia/reoxygenation to determine a transcriptomic CycHyp signature and used clinical data sets from 2,150 breast cancer patients to estimate a prognostic Cox proportional hazard model to assess its prognostic performance., Results: The CycHyp prognostic potential was validated in patients independently of the receptor status of the tumors. The discriminating capacity of the CycHyp signature was further increased in the ER+ HER2- patient populations including those with a node negative status under treatment (HR=3.16) or not (HR=5.54). The CycHyp prognostic signature outperformed a signature derived from continuous hypoxia and major prognostic metagenes (P<0.001). The CycHyp signature could also identify ER+HER2 node-negative breast cancer patients at high risk based on clinicopathologic criteria but who could have been spared from chemotherapy and inversely those patients classified at low risk based but who presented a negative outcome., Conclusions: The CycHyp signature is prognostic of breast cancer and offers a unique decision making tool to complement anatomopathologic evaluation.

Metastatic progression of cancer is associated with poor outcome, and here we examine metabolic changes underlying this process. Although aerobic glycolysis is known to promote metastasis, we have now identified a different switch primarily affecting mitochondria. The switch involves overload of the electron transport chain (ETC) with preserved mitochondrial functions but increased mitochondrial superoxide production. It provides a metastatic advantage phenocopied by partial ETC inhibition, another situation associated with enhanced superoxide production. Both cases involved protein tyrosine kinases Src and Pyk2 as downstream effectors. Thus, two different events, ETC overload and partial ETC inhibition, promote superoxide-dependent tumor cell migration, invasion, clonogenicity, and metastasis. Consequently, specific scavenging of mitochondrial superoxide with mitoTEMPO blocked tumor cell migration and prevented spontaneous tumor metastasis in murine and human tumor models., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

We hypothesized that vascular endothelial growth factor (VEGF)-containing hydrogels that gelify in situ after injection into a traumatized spinal cord, could stimulate spinal cord regeneration. Injectable hydrogels composed of 0.5% Pronova UPMVG MVG alginate, supplemented or not with fibrinogen, were used. The addition of fibrinogen to alginate had no effect on cell proliferation in vitro but supported neurite growth ex vivo. When injected into a rat spinal cord in a hemisection model, alginate supplemented with fibrinogen was well tolerated. The release of VEGF that was incorporated into the hydrogel was influenced by the VEGF formulation [encapsulated in microspheres or in nanoparticles or in solution (free)]. A combination of free VEGF and VEGF-loaded nanoparticles was mixed with alginate:fibrinogen and injected into the lesion of the spinal cord. Four weeks post injection, angiogenesis and neurite growth were increased compared to hydrogel alone. The local delivery of VEGF by injectable alginate:fibrinogen-based hydrogel induced some plasticity in the injured spinal cord involving fiber growth into the lesion site., (© 2013 Wiley Periodicals, Inc.)

High lactate concentration in tumors is associated with bad prognosis. Lactate is released by glycolytic cells in tumors and recaptured by oxidative cancer cells to feed the tricarboxylic acid (TCA) cycle after conversion into pyruvate. Monocarboxylate transporters (MCT) mediate these fluxes of proton-linked lactate and represent attractive targets to interrupt lactate shuttle and to inhibit tumor growth. Here, we investigated the properties of 7-aminocarboxycoumarins (7ACC) developed to selectively interfere with lactate fluxes in the lactate-rich tumor microenvironment. The pharmacologic properties of two compounds of this family, including their effects on lactate influx and efflux and antitumor activity, were investigated using human cancer cell lines and mouse xenograft models. Contrary to the reference MCT1 inhibitor AR-C155858, 7ACC unexpectedly inhibited lactate influx but not efflux in tumor cells expressing MCT1 and MCT4 transporters. 7ACC delayed the growth of cervix SiHa tumors, colorectal HCT116 tumors, and orthoptopic MCF-7 breast tumors. MCT target engagement was confirmed by the lack of activity of 7ACC on bladder UM-UC-3 carcinoma that does not express functional MCT. 7ACC also inhibited SiHa tumor relapse after treatment with cisplatin. Finally, we found that contrary to AR-C155858, 7ACC did not prevent the cell entry of the substrate-mimetic drug 3-bromopyruvate (3BP) through MCT1, and contributed to the inhibition of tumor relapse after 3BP treatment. In conclusion, our results indicate that 7ACC selectively affects a single part of the MCT symporter translocation cycle, leading to strict inhibition of lactate influx. This singular activity is associated with antitumor effects less prone to resistance and side effects., (©2014 American Association for Cancer Research.)

MRI cell tracking is a promising technique for tracking various cell types in living animals. Usually, cells are incubated with iron oxides so that the particles are taken up before the cells are injected in vivo. In the present study, we aimed to monitor migration of luciferase-expressing mouse renal cancer cells (RENCA-luc) after intrarenal or intrasplenic injection. These cells were labelled using Molday Ion Rhodamine B (MIRB) fluorescent superparamagnetic iron oxide particles. Their fate after injection was first assessed using ex vivo X-band electron paramagnetic resonance (EPR) spectroscopy. This biodistribution study showed that RENCA-luc cells quickly colonized the lungs and the liver after intrarenal and intrasplenic injection, respectively. Bioluminescence imaging (BLI) studies confirmed that this cell line preferentially metastasized to these organs. Early tracking of labelled RENCA-luc cells in the liver using high-field MRI (11.7 T) was not feasible because of a lack of sensitivity. MRI of MIRB-labelled RENCA-luc cells after injection in the left kidney was then performed. T2 - and T2 *-weighted images showed that the labelled cells induced hypointense signals at the injection site. Nevertheless, the hypointense regions tended to disappear after several days, mainly owing to dilution of the MIRB iron oxides with cell proliferation. In conclusion, EPR is well adapted to ex vivo analysis of tissues after cell tracking experiments and allows short-term monitoring of metastasizing cells. MRI is a suitable tool for checking labelled cells at their injection site, but dilution of the iron oxides owing to cell division remains a major limitation. BLI remains the most suitable technique for long-term monitoring of metastatic cells., (Copyright © 2014 John Wiley & Sons, Ltd.)

Aims: Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity, critical elements for their survival and differentiation, have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and, more generally, to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation., Methods and Results: MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes, a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand, A-769662, a well-characterized AMPK activator, was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice, providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation., Conclusion: MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation, p27 being involved in this regulation.

Under hypoxia, cancer cells consume glucose and release lactate at a high rate. Lactate was recently documented to be recaptured by oxygenated cancer cells to fuel the TCA cycle and thereby to support tumor growth. Monocarboxylate transporters (MCT) are the main lactate carriers and therefore represent potential therapeutic targets to limit cancer progression. In this study, we have developed and implemented a stepwise in vitro screening procedure on human cancer cells to identify new potent MCT inhibitors. Various 7-substituted carboxycoumarins and quinolinone derivatives were synthesized and pharmacologically evaluated. Most active compounds were obtained using a palladium-catalyzed Buchwald-Hartwig type coupling reaction, which proved to be a quick and efficient method to obtain aminocarboxycoumarin derivatives. Inhibition of lactate flux revealed that the most active compound 19 (IC50 11 nM) was three log orders more active than the CHC reference compound. Comparison with warfarin, a conventional anticoagulant coumarin, further showed that compound 19 did not influence the prothrombin time which, together with a good in vitro ADME profile, supports the potential of this new family of compounds to act as anticancer drugs through inhibition of lactate flux., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Easily measurable biomarkers are urgently required to detect early stages of cancer progression. Autoantibodies (aAbs), as a component of the humoral immune response against tumor cells, have such potential of diagnostic markers since they are circulating and stable proteins, produced rapidly and easily amenable to in vitro dosage. The identification of aAbs is based on the characterization of tumor-associated antigens (TAA) against which they are directed. Here, we propose a new method for an unbiased identification of TAA and thereby of aAbs as cancer biomarkers. This method that we called sequential immunoaffinity depletion-differential in gel electrophoresis (SID-DIGE) is based on the immunodepletion of tumor cell lysates with IgG from control and tumor-bearing mice and direct matching of the flow throughs of these immunoaffinity separations on the same 2D format. This strategy reduces the complexity of the samples to be analyzed and maximizes the interest of assessing hundreds of proteins simultaneously. SID-DIGE has also the potential, contrary to existing serological proteome analysis (SERPA) techniques, to detect immunogenic proteins with conformational epitopes, including those resulting from post-translational modifications. Using a model of human colorectal tumors in mice for the proof of principle, we showed that SID-DIGE outperforms the conventional SERPA technique, with the identification of 7 common TAA (validating our approach) and 18 additional aAbs proving the potential of this new method. In particular, the identification of aAbs directed against key enzymes supporting glycolysis gives credential to the role of hypoxia as a major determinant of the tumor proteome and thus as a source of immunogenicity. Overall, the developed methodology allowed efficient screening of sera for the identification of aAbs as potential biomarkers., (© 2013.)

The expression by tumor cells of proteins with aberrant structure, expression or distribution accounts for the development of a humoral immune response. Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer. Serological proteome analysis (SERPA) aims to identify such circulating aAb through the immunoblotting of 2D-separated tumor cell proteins with cancer patient serum and the consecutive MS identification of proteins in reactive spots. This method has the advantage to use post-translationally modified proteins as a source of potential TAA. Here, we applied this strategy by using colorectal tumor cells pre-exposed to hypoxia in order to promote the expression of a pattern of TAA more likely to represent in vivo conditions. We used two human HCT116 and HT29 colorectal cancer cell lines exposed for 48 hours to 1% O2. Spots positive after immunoblotting of 2D-separated lysates of hypoxic cells with the sera of tumor-bearing mice, were collected and analysed by MS for protein identification. Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2). We confirmed the increased phosphorylation of this protein in hypoxic colorectal tumor cells as well as in mouse tumors. Using a specific immunoassay, we could detect the presence of corresponding anti-phospho-Thr56 eEF2 aAb in the serum of tumor-bearing mice (vs healthy mice). We further documented that the detection of these aAb preceded the detection of a palpable tumor mass in mice and validated the presence of anti-phospho-Thr56 eEF2 aAb in the serum of patients with adenomatous polyps and colorectal carcinoma. In conclusion, this study validates a phosphorylated form of eEF2 as a new TAA and more generally, provides evidence that integrating hypoxia upstream of SERPA offers a more relevant repertoire of TAA able to unmask the presence of circulating aAb.

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by > 90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Antibody-labeled gold nanoparticles represent a promising novel tool regarding cancer imaging and therapy. Nevertheless, the characterization of biodistribution of such immunonanocarriers has been poorly documented. In this study, the biodistribution of (89)Zr-labeled cetuximab before and after the coupling reaction to gold nanoparticles (AuNPs) was compared and the quantitative imaging performance of (89)Zr immuno-PET was evaluated. Cetuximab was functionalized with the desferal moiety and labeled with (89)Zr ((89)Zr-Df-Bz-NCS-cetuximab). AuNPs with a mean diameter of 5 nm were synthesized according a new method developed in the laboratory, and conjugated to (89)Zr-Df-Bz-NCS-cetuximab using carbodiimide chemistry (AuNPs-PPAA-cetuximab-(89)Zr). The two tracers were injected in A431 xenograft-bearing mice. Tumor and liver uptakes were assessed at different times after injection using quantitative PET imaging. The in vivo specificity of the binding was investigated using a saturating dose of unlabeled cetuximab. Radiolabeled cetuximab was conjugated to AuNPs with a coupling reaction yield >75%. All conjugates were stable in vitro and to a lesser extent in plasma. In vivo distribution studies revealed no significant difference in tumor uptake for cetuximab conjugated to nanoparticles up to 72 h after injection, compared with unconjugated cetuximab. Immuno-PET studies showed that AuNPs-PPAA-cetuximab-(89)Zr provided high tumor-to-background ratio. The liver uptake of AuNPs-PPAA-cetuximab-(89)Zr was higher, compared with (89)Zr-Df-Bz-NCS-cetuximab. In vivo blocking experiments demonstrated selective tumor targeting after coupling reaction. This study showed that the conjugation of AuNPs to cetuximab did not affect its tumor accumulation and that the efficacy of EGFR-targeted nanoparticles was unaltered. The (89)Zr-labeled cetuximab-targeted gold nanoparticles could be a valuable tool for theranostic purposes., (Copyright © 2013 John Wiley & Sons, Ltd.)

Purpose: Because of its paramagnetic properties, oxygen may act as an endogenous magnetic resonance imaging contrast agent by changing proton relaxation rates. Changes in tissue oxygen concentrations have been shown to produce changes in relaxation rate R1 of water. The aim of the study was to improve the sensitivity of oxygen enhanced R1 imaging by exploiting the higher solubility of oxygen in lipids (as compared with water) to sensitively monitor changes in tissue oxygen levels by selectively measuring the R1 of lipids., Methods: The method, with the acronym "MOBILE" (mapping of oxygen by imaging lipids relaxation enhancement), was applied in different mouse models of hypoxic processes on a 11.7 T magnetic resonance imaging system. MOBILE was compared with R*2, R1 of water, and with pO2 measurements (using electron paramagnetic resonance oximetry). MOBILE was also applied in the brain of healthy human volunteers exposed to an oxygen breathing challenge on a 3 T magnetic resonance imaging system., Results: MOBILE was shown to be able to monitor changes in oxygenation in tumor, peripheral, liver, and brain tissues. The clinical translation was demonstrated in human volunteers., Conclusion: MOBILE arises as a promising noninvasive and sensitive tool for diagnosis and therapeutic guidance in disorders involving hypoxia., (Copyright © 2012 Wiley Periodicals, Inc.)

Background and Purpose: We hypothesized that electrotransfer of a plasmid encoding an antiangiogenic factor, the recombinant disintegrin domain of ADAM-15, (pRDD) could modify the tumor microenvironment and radiosensitize tumor., Materials and Methods: pRDD was injected in the TLT tumor or FSaII fibrosarcomas before electroporation. pO2 in tumors and oxygen consumption in vitro were measured by electronic paramagnetic resonance (EPR) oximetry. Tumor perfusion was assessed by laser doppler imaging and patent blue assay., Results: pRDD electrotransfer caused a significant delay in TLT growth and an anti-angiogenic effect. It significantly increased tumor pO2 in TLT and FSaII for at least 4 days. pRDD electrotransfer and radiotherapy were more effective than either treatment alone. Modifications of tumor microenvironment were evaluated: tumor perfusion and interstitial fluid pressure were not modified. Oxygen consumption by the cells was decreased resulting both from a decrease in oxygen consumption rate and from a decrease in cell viability., Conclusion: The combination of localized antiangiogenic gene therapy and radiotherapy applied in the time of maximal oxygenation could be a promising alternative for cancer treatment., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The purpose of this study was to determine the value of different imaging modalities, that is, magnetic resonance imaging/spectroscopy (MRI/MRS) and positron emission tomography (PET), to assess early tumor response to sorafenib with or without radiotherapy. Diffusion-weighted (DW)-MRI, choline (1)H MRS at 11.7 T, and (18)F-FLT PET imaging were used to image fibrosarcoma (FSaII) tumor-bearing mice over time. The imaging markers were compared with apoptosis cell death and cell proliferation measurements assessed by histology. Anti-proliferative effects of sorafenib were evidenced by (1)H MRS and (18)F-FLT PET after 2 days of treatment with sorafenib, with no additional effect of the combination with radiation therapy, results that are in agreement with Ki67 staining. Apparent diffusion coefficient calculated using DW-MRI was not modified after 2 days of treatment with sorafenib, but showed significant increase 24 h after 2 days of sorafenib treatment combined with consecutive irradiation. The three imaging markers were able to show early tumor response as soon as 24 h after treatment initiation, with choline MRS and (18)F-FLT being sensitive to sorafenib in monotherapy as well as in combined therapy with irradiation, whereas DW-MRI was only sensitive to the combination of sorafenib with radiotherapy., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK., Methods: The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides., Results: Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation., Conclusions: Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.

Glioblastoma multiforme (GBM) is a deadly cancer that possesses an intrinsic resistance to pro-apoptotic insults, such as conventional chemotherapy and radiotherapy, and diffusely invades the brain parenchyma, which renders it elusive to total surgical resection. We found that fusicoccin A, a fungal metabolite from Fusicoccum amygdali, decreased the proliferation and migration of human GBM cell lines in vitro, including several cell lines that exhibit varying degrees of resistance to pro-apoptotic stimuli. The data demonstrate that fusicoccin A inhibits GBM cell proliferation by decreasing growth rates and increasing the duration of cell division and also decreases two-dimensional (measured by quantitative video microscopy) and three-dimensional (measured by Boyden chamber assays) migration. These effects of fusicoccin A treatment translated into structural changes in actin cytoskeletal organization and a loss of GBM cell adhesion. Therefore, fusicoccin A exerts cytostatic effects but low cytotoxic effects (as demonstrated by flow cytometry). These cytostatic effects can partly be explained by the fact that fusicoccin inhibits the activities of a dozen kinases, including focal adhesion kinase (FAK), that have been implicated in cell proliferation and migration. Overexpression of FAK, a nonreceptor protein tyrosine kinase, directly correlates with the invasive phenotype of aggressive human gliomas because FAK promotes cell proliferation and migration. Fusicoccin A led to the down-regulation of FAK tyrosine phosphorylation, which occurred in both normoxic and hypoxic GBM cell culture conditions. In conclusion, the current study identifies a novel compound that could be used as a chemical template for generating cytostatic compounds designed to combat GBM.

Background and Purpose: Tumours are now considered as complex tissues including endothelial cells of the tumour vasculature, which can decrease radiotherapy efficacy. It is thus important to better characterise the response of both types of cells to irradiation. This study investigated the effects of X-ray and alpha particle irradiation on cancer and endothelial cells., Materials and Methods: A549 non-small-cell lung adenocarcinoma cells and human endothelial cells (EC) were exposed to X-rays or alpha particles. Responses were studied by clonogenic assays and nuclei staining. A gene expression study was performed by using Taqman low density array and the results were validated by qRT-PCR and ELISA., Results: The relative biological effectiveness of alpha particles was estimated to be 5.5 and 4.6 for 10% survival of A549 cells and EC, respectively. Nuclei staining indicated that mitotic catastrophe was the main type of cell death induced by X-rays and alpha particles. Both ionising radiations induced the overexpression of genes involved in cell growth, inflammation and angiogenesis., Conclusions: Alpha particle irradiations are more effective than X-rays. The gene expression changes observed in both cell types after alpha particle or X-ray exposure showed possible crosstalk between both cell types that may induce the development of radioresistance., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study.

Wounds notoriously accumulate lactate as a consequence of both anaerobic and aerobic glycolysis following microcirculation disruption, immune activation, and increased cell proliferation. Several pieces of evidence suggest that lactate actively participates in the healing process through the activation of several molecular pathways that collectively promote angiogenesis. Lactate indeed stimulates endothelial cell migration and tube formation in vitro, as well as the recruitment of circulating vascular progenitor cells and vascular morphogenesis in vivo. In this study, we examined whether the pro-angiogenic potential of lactate may be exploited therapeutically to accelerate wound healing. We show that lactate delivered from a Matrigel matrix improves reperfusion and opposes muscular atrophy in ischemic hindlimb wounds in mice. Both responses involve lactate-induced reparative angiogenesis. Using microdialysis and enzymatic measurements, we found that, contrary to poly-L-lactide (PLA), a subcutaneous implant of poly-D,L-lactide-co-glycolide (PLGA) allows sustained local and systemic lactate release. PLGA promoted angiogenesis and accelerated the closure of excisional skin wounds in different mouse strains. This polymer is FDA-approved for other applications, emphasizing the possibility of exploiting PLGA therapeutically to improve wound healing.

Background: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut., Methods: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and qPCR. Serum Short-chain fatty acids were quantified by UHPLC-MS. Cell proliferation was evaluated in vivo, by molecular biology and histology, and in vitro., Results: Inulin-type fructans treatment reduces hepatic BaF3 cell infiltration, lessens inflammation and increases portal propionate concentration. In vitro, propionate reduces BaF3 cell growth through a cAMP level-dependent pathway. Furthermore, the activation of free fatty acid receptor 2 (FFA2), a Gi/Gq-protein-coupled receptor also known as GPR43 and that binds propionate, lessens the proliferation of BaF3 and other human cancer cell lines., Conclusion: We show for the first time that the fermentation of nutrients such as ITF into propionate can counteract malignant cell proliferation in the liver tissue. Our results support the interest of FFA2 activation as a new strategy for cancer therapeutics. This study highlights the importance of research focusing on gut microbes-host interactions for managing systemic and severe diseases such as leukaemia.

Background: This phase II study assessed the safety and efficacy of everolimus, an oral mammalian target of rapamycin inhibitor in advanced transitional carcinoma cell (TCC) after failure of platinum-based therapy., Patients and Methods: Thirty-seven patients with advanced TCC received everolimus 10 mg/day until progressive disease (PD) or unacceptable toxicity. The primary end point was the disease control rate (DCR), defined as either stable disease (SD), partial response (PR), or complete response at 8 weeks. Angiogenesis-related proteins were detected in plasma and changes during everolimus treatment were analyzed. PTEN expression and PIK3CA mutations were correlated to disease control., Results: Two confirmed PR and eight SD were observed, resulting in a DCR of 27% at 8 weeks. Everolimus was well tolerated. Compared with patients with noncontrolled disease, we observed in patients with controlled disease a significant higher baseline level of angiopoietin-1 and a significant early plasma decrease in angiopoietin-1, endoglin, and platelet-derived growth factor-AB. PTEN loss was observed only in patients with PD., Conclusions: Everolimus showed clinical activity in advanced TCC. The profile of the plasma angiogenesis-related proteins suggested a role of the everolimus antiangiogenic properties in disease control. PTEN loss might be associated with everolimus resistance.

Background and Purpose: The relevance of Mitogen Activated Protein Kinase (MAPK) inhibitors as co-treatments for radiation therapy is investigated, with special focus on a potential link between the MAPK pathway and tumor hypoxia, which is a critical determinant for response to therapy., Materials and Methods: The effects of two MAPK inhibitors, Sorafenib and PD0325901, were monitored daily using in vivo EPR (Electron Paramagnetic Resonance) oximetry in FSaII and TLT tumor models in order to identify a window of reoxygenation, during which tumor blood flow, oxygen consumption and radiation sensitivity were assessed., Results: Reoxygenation was shown after two days of treatments with Sorafenib or PD0325901 in two tumor models, which was further successfully exploited with Sorafenib for improving the radiation response of FSaII tumors by a factor of 1.5. The increase in tumor oxygenation was shown to be the result of two major factors: (i) an increase in blood flow for Sorafenib, that might be linked to its anti-angiogenic effect (vascular normalization), and (ii) a decrease in oxygen consumption for Sorafenib and PD0325901, due to an alteration of the mitochondrial activity., Conclusion: We evidenced tumor reoxygenation in vivo following MAPK inhibition and suggest a rationale for the combination of radiation therapy with Sorafenib., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Aggressiveness of advanced melanomas relates in part to their marked propensity to develop neoangiogenesis and metastases. Among its numerous pro-cancer roles, galectin (gal)-1 expressed and/or secreted by both cancer and endothelial cells stimulates proliferation and angiogenesis. This study first shows that gal-1 is more highly expressed at both mRNA and protein levels than its congeners in melanomas and particularly in advanced lesions. The roles of gal-1 were further investigated in vivo in the highly proliferating and vascularized pseudometastatic B16F10 mouse melanoma model using stable knockdown B16F10 cells and wild-type versus gal-1 knockout mice, and then in vitro in B16F10 tumoral and lung microvascular cells. Gal-1 depletion in the B16F10 tumor cells but not in the tumor-bearing mice significantly increased melanoma-bearing mice survival. Tumor-derived gal-1 thus seems to have more critical roles than the host-derived one. In fact, gal-1 displays distinct effects on the H-Ras-dependent p53/p21 pathways: in primary lung microvessel endothelial cells, gal-1 seems to be involved in the maintenance of senescent status through the induction of both p53 and p21 while it stimulates B16F10 cancer cell proliferation through a p53/p21 decrease. Altogether, these data point to gal-1 as a potential target to combat melanomas.

Objective: We investigated the impact of hypoxia-reoxygenation on endothelial relaxation and aimed to clarify the role of transient receptor potential cation channels V4 (TRPV4) and gap junctions in the protective effect associated with hypoxic preconditioning on the vascular function., Methods and Results: By mimicking ischemia-reperfusion in C57BL/6 male mice in vivo, we documented a reduced NO-mediated relaxation and an increased endothelium-derived hyperpolarization (EDH[F])-mediated relaxation. Hypoxic preconditioning, however, restored NO relaxation and further improved the EDH(F) response. We also examined specifically 2 major effectors of the EDH(F) pathway, transient receptor potential cation channels V4 and connexins. We found that in endothelial cells, expression and activity of transient receptor potential cation channels V4 were increased by hypoxic stimuli independently of preconditioning which was interestingly associated with an increase of structural caveolar component caveolin-1 at membrane locations. Gap junctions, however, seemed to directly support EDH(F)-driven preconditioning as connexin 40 and connexin 43 expression increased and as in vivo carbenoxolone treatment completely inhibited the EDH(F) pathway and significantly reduced the protection afforded by preconditioning for the concomitant NO-mediated relaxation., Conclusions: Our work provides evidence on how transient receptor potential cation channels V4 and connexins might participate in preserving vasorelaxation under hypoxia and restoring the NO-mediated pathway in hypoxic preconditioning conditions pointing out caveolae as a common signaling location.

MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perls's Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results., (Copyright © 2012 John Wiley & Sons, Ltd.)

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations., (Copyright © 2011 John Wiley & Sons, Ltd.)

Background: The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated., Methods: We investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments., Results: The N-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and N- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor., Conclusions: This study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.

Purpose: Lung cancer is the leading cause of cancer-related death. Among the new modalities to treat cancer, internal radiotherapy seems to be very promising. However, the achievable dose-rate is two orders of magnitude lower than the one used in conventional external radiotherapy, and data has to be collected to evaluate the cell response to highlight the potential effectiveness of low-dose-rate beta particles irradiation. This work investigates the phosphorus beta irradiation ((32)P) dose response on the clonogenicity of human A549 non-small cell lung adenocarcinoma cells and compares it to high-dose-rate X-irradiations results., Materials and Methods: Cell survival was evaluated by a colony forming assay eight days after low-dose-rate (32)P beta irradiations (0.8 Gy/h) and high-dose-rate X-ray irradiations (0.855 Gy/min)., Results: Survival curves were obtained for both types of irradiations, and showed hyper-radiosensitivity at very low doses. Radiosensitivity parameters were obtained by using the linear-quadratic and induced-repair models., Conclusions: Comparison with high-dose-rate X-rays shows a similar surviving fraction, confirming the effectiveness of beta particles for tumor sterilization.

The monocarboxylate transporter (MCT) family member MCT1 can transport lactate into and out of tumor cells. Whereas most oxidative cancer cells import lactate through MCT1 to fuel mitochondrial respiration, the role of MCT1 in glycolysis-derived lactate efflux remains less clear. In this study, we identified a direct link between p53 function and MCT1 expression. Under hypoxic conditions, p53 loss promoted MCT1 expression and lactate export produced by elevated glycolytic flux, both in vitro and in vivo. p53 interacted directly with the MCT1 gene promoter and altered MCT1 mRNA stabilization. In hypoxic p53(-/-) tumor cells, NF-κB further supported expression of MCT1 to elevate its levels. Following glucose deprivation, upregulated MCT1 in p53(-/-) cells promoted lactate import and favored cell proliferation by fuelling mitochondrial respiration. We also found that MCT1 expression was increased in human breast tumors harboring p53 mutations and coincident features of hypoxia, with higher MCT1 levels associated with poorer clinical outcomes. Together, our findings identify MCT1 as a target for p53 repression and they suggest that MCT1 elevation in p53-deficient tumors allows them to adapt to metabolic needs by facilitating lactate export or import depending on the glucose availability.

Arsenic trioxide (As(2)O(3)) is an effective therapeutic against acute promyelocytic leukemia and certain solid tumors. Because As(2)O(3) inhibits mitochondrial respiration in leukemia cells, we hypothesized that As(2)O(3) might enhance the radiosensitivity of solid tumors by increasing tumor oxygenation [partial pressure of oxygen (pO(2))] via a decrease in oxygen consumption. Two murine models of radioresistant hypoxic cancer were used to study the effects of As(2)O(3). We measured pO(2) and the oxygen consumption rate in vivo by electron paramagnetic resonance oximetry and (19)fluorine-MRI relaxometry. Tumor perfusion was assessed by Patent blue staining. In both models, As(2)O(3) inhibited mitochondrial respiration, leading to a rapid increase in pO(2). The decrease in oxygen consumption could be explained by an observed decrease in glutathione in As(2)O(3)-treated cells, as this could increase intracellular reactive oxygen species that can disrupt mitochondrial membrane potential. When tumors were irradiated during periods of As(2)O(3)-induced augmented oxygenation, radiosensitivity increased by 2.2-fold compared with control mice. Notably, this effect was abolished when temporarily clamped tumors were irradiated. Together, our findings show that As(2)O(3) acutely increases oxygen consumption and radiosensitizes tumors, providing a new rationale for clinical investigations of As(2)O(3) in irradiation protocols to treat solid tumors.

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities.